Major depressive disorder is a leading cause of disability worldwide, affecting up to 17 % of the general population. The neural mechanisms of depression, however, are yet to be uncovered. Recently, attention has been drawn to the effects of dysfunctional brain-gut axis on depression, and many substances have been suggested to be involved in the communication between the gut and brain, such as ghrelin.We herein systematically examined the changes of metabolomics after unpredictable chronic mild stress (UCMS)–induced depression-like behaviors in rats and compared the altered metabolites in the hippocampus and jejunum samples.Our results show that many metabolites significantly changed with UCMS both in the hippocampus and jejunum, such as L-glutamine, L-tyrosine, hydroxylamine, and 3-phosphoglyceric acid. Further studies suggested that these changes are the reasons for anxiety-like behaviors and depression-like behaviors in UCMS rats and also are the reasons for hippocampal neural plasticity.Coexistence of brain and gut metabolic changes in UCMS-induced depressive behavior in rats suggests a possible role of brain-gut axis in depression. This study provides insights into the neurobiology of depression.The online version contains supplementary material available at 10.1007/s00213-021-05958-w. Major depression is affecting more than 17% of populations worldwide  tract, can modulate the activities of the central nervous system. For example, it is found that transplantation of microbiota from depressed patients to germ-free rats led to depressive behaviors . N-hexane was purchased from ROE Scientific . Methanol of MS grade was supplied by Merck Millipore . Ultrahigh-purity water was prepared by Millipore-Q system .Methoxyamine hydrochloride, pyridine, N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS), fluoxetine, and 1,2-−1). Group III started from the third week and were given continuous intragastric drug administration for 3 weeks continuously, 3 times a day. Fluoxetine solution was dissolved in 0.9 % saline  and unpredictable chronic mild stress (UCMS) models to evaluate the susceptibility versus resilient phenotypes in male and female rats. SCVS induced depression-like behaviors in female rats only. The UCMS paradigm was more likely to induce depression-like behaviors in male rats  which was performed on day 35, open field test (OFT) on day 36, and forced swim test (FST) on day 37, as we previously reported . Immediately, thereafter, 50 μL of BSTFA trifluoroacetamide (BSTFA) containing 1 % TMS was added, and the sample was shaken again at 37 °C for 0.5 h. The mixture was then transferred to a sampler vial and subjected to GC-MS analysis.Sample preparation for GC-MS analysis was revised according to previous reports  was added for vortexing for 3 min to precipitate the protein and mix the internal standard with the sample. The mixture was centrifuged at 27400 × g for 10 min at 4 °C, and 300 μL of 300 supernatants was dried at 45 °C and 15 kPa to evaporate MeOH. After drying, the sample was added to 50 μL of a concentration of 15-mg/mL methoxyamine pyridine and vortexed for 5 min, and then the sample was reconstituted by shaking at 30°C for 90 min with the ThermoMixer C . Immediately after, 60 μL of BSTFA trifluoroacetamide containing 1% TMS was added, vortexed for 1 min, and shaken again at 37 °C for 0.5 h. Subsequently, 60 μL of the supernatant was centrifuged at 45300 × g for 10 min and then injected into the GC-MS for analysis. All derivatization must be carried out in an anhydrous environment. Quality control (QC) samples of the hippocampus and jejunum were also prepared and derivatized according to the abovementioned protocol and were run after every 12 samples to monitor the retention time and elution order of the metabolites.We chose jejunum to represent gut samples for analysis according to previous reports. The jejunum begins with the duodenal jejunum curve, especially the upper third is the most abundant part of the folds, the most important part of nutrient absorption, and the most important part of blood supply. Changes in jejunum mRNA expression can trigger neuromodulation and endocrine-related pathways and play a key role in metabolic-related diseases .Metabolomics analysis of hippocampus and jejunum samples was performed using a Thermo Trace 1310-TSQ 8000 gas chromatograph system coupled to a mass spectrometer. Each 1 μL of the derivative sample was injected onto a TG-5MS capillary column , and the excitation mode was separated by a ratio of 20:1. The gradient heating used helium (99.999%) as a carrier gas, which was maintained at a constant flow rate of 1.2 mL/min. GC temperature changes are as follows: 0–1 min, 60 °C; 1–14 min, 60–320 °C; and 14–19 min, 320 °C. The electron energy is 70 eV, and the MS data were obtained in full scan mode with a mass range of 50–500 m/z and a time range of 3.5–19 min. The QC was randomized by sequence, and a QC sample was injected between every 8 actual sample to check the peak intensity of the spiked internal standard , as previously reported . And after the perfusion, the brain was decapitated immediately and was placed in ice-cold aCSF for 2 min. aCSF composition was 125 mM NaCl, 3.25 mM KCl, 1.25 mM NaHThe whole perfusion system in vitro was continuously perfused with normal aCSF with 95 % oxygen and 5 % carbon dioxide. The temperature of cerebrospinal fluid was maintained at 24 °C and the flow rate was about 2–3 mL/min. A borate glass microelectrode filled with aCSF was used to record the evoked excitatory postsynaptic potentials. The concentric bipolar electrode  was placed in the Schaffer collateral branch to deliver presynaptic stimulation. The glass microelectrode was placed in the stratum radiatum of the CA1 area. The fEPSP corresponding to 30%–40% of the maximum amplitude response is tested. The stimulation electrode was used to deliver the test stimulus every 30 s, with each stimulus lasted 100 μs. After recording the stable baseline for at least 10 min, the high frequency stimulus program  was applied, and LTP was induced. LTP was recorded with the same baseline stimulation intensity  and recorded for at least 60 min. The slope of the induced fEPSP was measured and normalized to an average baseline of one minute. The field potential recording was filtered at 2 kHz, sampled at 20 kHz, and continuously recorded using a MultiClamp 700B amplifier , and electrophysiological data was analyzed with Clampfit 10.5 software (Molecular Devices). The group allocations of the rats were blinded to the experimenters.t-test. All statistical analyses were carried out using SPSS software . A p value less than 0.05 (p<0.05) was considered statistically significant. GC-MS data (Raw Data Files) of all samples were converted using the ABF converter  into MS-DIAL (v.2.7.2) software program  before a series of multivariate statistical analyses, and the statistical significance of the identified metabolites was assessed using one-way ANOVA and q-test. The direct threshold changes of the final metabolite > 1.2 and p< 0.05 can be considered as potentially altered metabolites. All statistical analyses were then plotted using Prism 8 (GraphPad 8.00) and MetaboAnalyst 4.0.All data are shown as the mean ± SEM. Differences in behavioral results were evaluated using Student’s p < 0.01, compared with UCMS group; p < 0.01, compared with control group, one-way ANOVA, n=12, Fig. p < 0.01 vs. control, one-way ANOVA, n=12, Fig. p < 0.05), indicating that depression was successfully induced. In addition, we also monitored body weight and plasma corticosterone (CORT) over the UCMS procedure. Our results showed that UCMS significantly decreased body weight and increased the CORT, which is consistent with our previous reports  rats were used to investigate the effects of chronic stress on the metabolic changes in both brain and gut. To determine if UCMS could successfully induce depression-like behaviors, we measured behavior changes, body weight, and plasma corticosterone (CORT) concentration of the rats after the UCMS training. Sucrose preference test, forced swim test (FST), and open field test (OFT) were used to test UCMS-induced depression-like behaviors. The results showed that UCMS significantly decreased the sucrose consumption in the sucrose preference test (SPT)  of the hippocampus Fig. . In hippp< 0.05, t-test, n=8, Fig. p< 0.05, t-test, n=8, Fig. 13C2-myristic acid was 13.64 % for the hippocampus and 7.58 % for the jejunum, indicating that the analytical conditions and sample process exhibited good repeatability and stability for the metabolomic study.System stability and reproducibility analysis in both hippocampus and jejunum samples were performed based on GC-MS to obtain biochemical information on metabolites. The multivariate analysis method of principal component analysis (PCA) was done to investigate the similarities and differences among the groups. The PCA score scatter plot Fig.  demonstrq-test (p < 0.05), and there was a fold change >1.2 between the control and UCMS groups. The details of the altered metabolites in the hippocampus and jejunum samples are presented in Table There were 48 potential biomarkers in the hippocampus and in the jejunum that significantly changed by UCMS Table , based oThe quantification of common metabolites in the hippocampus and jejunum samples in UCMS-induced depression-like behavior in rats were analyzed Fig. . CompareIn order to verify the correlation between the jejunum and hippocampus, the metabolite profile of serum samples was also studied with GC-MS. A total of 101 endogenous metabolites were identified in serum samples. After the data was normalized, QC was used to test the stability of the samples Fig. , and thep < 0.01, n=4).We recorded LTP in CA1 region of the hippocampal slices of UCMS group rats and control group rats. LTP was successfully inducted and recorded Fig.  in normaIn this study, we performed a comprehensive analysis of metabolic changes in brain-gut axis of rats with UCMS-induced depression-like behavior. Our data show that UCMS-induced depression-like behavior was accompanied with many metabolic changes that similarly happened in both the hippocampus and intestine, such as hydroxylamine, L-glutamine, L-tyrosine, and 3-phosphoglyceric acid. These similar metabolite changes in the jejunum and hippocampus confirm that the brain-gut axis is closely related to the pathogenesis of depression-like behavior.http://www.kegg.jp/), and Human Metabolome Database (HMDB) (http://www.hmdb.ca/), we conducted metabolic pathway analysis based on the distribution of differential metabolites, and a network of pathways of brain-gut axis metabolism in rats with UCMS-induced depression-like behavior was built with Pathway Builder Tool 2.0  derived from hydroxylamine and S-nitroso-L-cysteine (NO-CYS) can evoke release of hippocampal NE ESM 2(PDF 783 kb)


Praziquantel (PZQ) is currently the only recommended drug for infection and disease caused by the schistosome species that infects humans; however, the current tablet formulation is not suitable for pre-school age children mainly due to its bitterness and the large tablet size. We assessed the palatability of two new orally disintegrating tablet (ODT) formulations of PZQ.t = 0 primary outcome) and after 2–5 minutes (VASt = 2–5).This randomized, single-blind, crossover, swill-and-spit palatability study (NCT02315352) was carried out at a single school in Tanzania in children aged 6–11 years old, with or without schistosomiasis infection as this was not part of the assessment. Children were stratified according to age group (6–8 years or 9–11 years) and gender, then randomized to receive each formulation in a pre-specified sequence. Over 2 days, the children assessed the palatability of Levo-Praziquantel (L-PZQ) ODT 150 mg and Racemate Praziquantel (Rac-PZQ) ODT 150 mg disintegrated in the mouth without water on the first day, and L-PZQ and Rac-PZQ dispersed in water and the currently available PZQ 600 mg formulation (PZQ-Cesol) crushed and dispersed in water on the second day. The palatability of each formulation was rated using a 100 mm visual analogue scale (VAS) incorporating a 5-point hedonic scale, immediately after spitting out the test product (VASt = 0 between the two ODT formulations (p = 0.106) without water. Higher VASt = 0 and VASt = 2–5 scores were reported for L-PZQ ODT compared with Rac-PZQ ODT in older children . The VASt = 0 and VASt = 2–5 were higher for both ODT formulations compared with the standard formulation (p<0.001 for both time points). No serious adverse events were reported.In total, 48 children took part in the assessment. Overall, there was no reported difference in the VASThe new paediatric-friendly formulations dispersed in water were both found to be more palatable than the existing standard formulation of PZQ. There may be gender and age effects on the assessment of palatability. Further research is needed for assessing efficacy and tolerability of the newly ODTs Praziquantel drug in younger children.NCT02315352) and in the Pan African Clinical Trials Registry (PACTR201412000959159).The trial was registered on ClinicalTrials.gov  to assess the new orally disintegrating isomers of Praziquantel, L-PZQ and Rac-PZQ which have been prepared as 150mg tablets and sweetened through the addition of a sweetener , in comparision to the existing Praziquantel formulation. Findings from 48 African children showed that both new formulations are more palatable to younger children as compared to the existing Praziquantel formulation.These results provided confidence for further evaluation of the clinical efficacy and tolerability of the paediatric friendly formulations of Praziquantel tablets for schistosomiasis treatment. Schistosoma that infect humans are Schistosoma haematobium, which causes urogenital schistosomiasis, as well as Schistosoma japonicum and Schistosoma mansoni, which cause intestinal schistosomiasis) or median  for quantitative variables. Descriptive analyses of outcome variables were stratified by formulation/preparation group and time of assessment. The VAS was summarized using descriptive statistics according to the formulation/preparation group.A1), as opposed to the null hypothesis (H01) that the tastes of the two formulations are indistinguishable. This was assessed using a linear mixed model with a factor variable for the two formulations and with random intercepts at the level of the children. The second hypothesis (HA2) was that at least one of the two new formulations (with water) differs in taste from the current PZQ-Cesol formulation. Here, each of the new formulations was tested against the current formulation at alpha = 0.025. The test sequence, gender, and age of the participants were considered when testing these hypotheses.To test the palatability, it was hypothesized that Rac-PZQ ODT (without water) and L-PZQ ODT (without water) taste different . The model with the lowest AIC value that included at least the independent factors  was selected as the final model. P-values were considered significant at the two-sided alpha = 0.05 level with no adjustment for multiple comparisons across models. The analysis was completed using R software During the screening, 75 children who had undergone repeated training rounds on how to adhere to study procedures were identified. Of these, 48 children were deemed eligible for participation distributed in equal-sized groups according to age group and gender and randomized such that equal numbers received each test sequence in each group. An additional 3 eligible participants were identified as reserve participants in case one of the first 48 participants withdrew but were not used. The weight of the participants ranged from 14.7 to 33.1 kg, corresponding to PZQ doses of 4.5 to 10.2 mg/kg if the 150 mg dose were to be unintentionally swallowed during the process. Mean (SD) body temperature, respiratory rates and heart rates were within normal ranges before and after each assessment .t = 0 scores in millimeters (both age groups and genders combined) for L-PZQ ODT and Rac-PZQ ODT without water were 49.0 (33.7) and 39.3 (28.4), respectively. The mean difference (SD) in VASt = 0 scores between the two formulations was not significant   VAS= 0.106) .t = 2–5 scores for L-PZQ ODT and Rac-PZQ ODT without water were 50.7 (27.2) and 45.1 (29.6), respectively. The overall mean (SD) difference in VASt = 2–5 scores between the two formulations was not significant . However, there was a significant interaction between the formulation and age group at this time point (interaction p = 0.0499). The mean VAS scores for L-PZQ ODT without water did not differ from scores for Rac-PZQ ODT without water in younger children (6–8 years old). Among older children (9–11 years old), VAS scores for L-PZQ ODT without water compared with Rac-PZQ ODT without water were higher by more than 15 mm   VASctively) . The disant  mm, while for L-PZQ ODT dispersed in water it was 67.5 (31.1) mm and for Rac-PZQ ODT dispersed in water it was 51.4 (32.4) mm. Similarly, at 2–5 minutes, the overall mean (SD) VASt = 2–5 scores were 33.5 (22.2) for current PZQ-Cesol, 61.1 (21.2) for L-PZQ ODT and 50.2 (25.9) for Rac-PZQ ODT in water. The differences in VAS scores for L-PZQ ODT and Rac-PZQ ODT dispersed in water compared with PZQ-Cesol were higher at both time points  VASp<0.001; . Besides p<0.01; . The dist = 0 and VASt = 2–5 for L-PZQ ODT dispersed in water were significantly higher than for Rac-PZQ ODT dispersed in water  participants reported occurrences of discomfort each during the study. One child reported abdominal discomfort on Day 2; this child had the Day 2 sequence of Rac-PZQ ODT/current PZQ-Cesol/L-PZQ ODT. Another child reported a headache on Day 2; this child had the Day 2 sequence of Rac-PZQ ODT/L-PZQ ODT/PZQ-Cesol. One child (2.1%) presented with malaria, which was constitutionally considered an adverse event before tasting on Day 2. This child was removed from the study and treated with appropriate antimalarial medication; she was followed up and was not replaced in the study with another child.When the two ODT formulations were tasted without water, mean VAS scores were acceptable in the “Neither good nor bad” range of the hedonic VAS scale and the VAS score for L-PZQ ODT did not differ from that for Rac-PZQ ODT. Overall, the between-participant variability was considerable, especially for age and gender. Although limited to relatively small sample sizes of 24 participants each, analyses stratified for the two age groups revealed that L-PZQ ODT without water was significantly more palatable than Rac-PZQ ODT without water in children aged between 9 and 11 years. These results, therefore, provide some evidence for the better palatability of L-PZQ ODT without water over Rac-PZQ ODT without water in older children. This was not observed in younger children. The age differences remain an important factor to accurately report on the palatability of each preparation without water.When tasted with water, both ODT formulations were more palatable than the current PZQ-Cesol crushed in water. Like without water, the mean VAS scores of the ODT formulations were in the “Neither good nor bad” range while the PZQ-Cesol mean VAS scores were in the “Poor Taste” part of the hedonic VAS scale. Palatability reported immediately after spitting out the IMP by boys differed from what was reported by girls; boys reported higher for L-PZQ ODT and lower for PZQ-Cesol. Although limited to relatively small sample sizes of 24 boys and 23 girls, analyses stratified by gender still revealed significantly higher scores for both the new formulations dispersed in water compared with the current PZQ-Cesol formulation. It is of note that L-PZQ ODT dispersed in water was more palatable compared with Rac-PZQ ODT dispersed in water at both time points for boys but not for girls. Thus, gender continues to be an important factor along with age to consider when reporting palatability. Another observation was the effect size for the difference between two ODTs formulations was a bit fairly large and the P-value was about 0.1  which liThis study was limited to children aged 6 years and older; pre-school children  were notIn conclusion, this trial has shown that the overall palatability of both L-PZQ ODT and Rac-PZQ ODT was higher than that of standard PZQ-Cesol when the drugs were dispersed in water in this study population of African children. The superiority of L-PZQ ODT over Rac-PZQ ODT without water could not be confirmed; however, L-PZQ ODT dispersed in water was more palatable than Rac-PZQ ODT dispersed in water. Some evidence supporting an age effect and gender effect on palatability was also observed.S1 File(PDF)Click here for additional data file.S2 File(DOC)Click here for additional data file.


Curcuma Longa has been used in therapeutic preparations for centuries in different parts of the world. However, its bioactivity is limited by chemical instability, water insolubility, low bioavailability, and extensive metabolism. In this study, the coaxial electrospinning technique was used to produce both poly (ε-caprolactone) (PCL)–curcumin and core–shell nanofibers composed of PCL and curcumin in the core and poly (lactic acid) (PLA) in the shell. Morphology and physical properties, as well as the release of curcumin were studied and compared with neat PCL, showing the formation of randomly oriented, defect-free cylindrical fibers with a narrow distribution of the dimensions. The antibacterial and antibiofilm potential, including the capacity to interfere with the quorum-sensing mechanism, was evaluated on Pseudomonas aeruginosa PAO1, and Streptococcus mutans, two opportunistic pathogenic bacteria frequently associated with infections. The reported results demonstrated the ability of the Curcumin-loading membranes to inhibit both PAO1 and S. mutans biofilm growth and activity, thus representing a promising solution for the prevention of biofilm-associated infections. Moreover, the high biocompatibility and the ability to control the oxidative stress of damaged tissue, make the synthesized membranes useful as scaffolds in tissue engineering regeneration, helping to accelerate the healing process.Curcumin extracted from the rhizome of  Curcuma Longa L., is well known for its beneficial effect on human health because of its outstanding anti-inflammatory [Among natural compounds, curcumin -1,6-heptadiene-3,5-dione), the main active components isolated from the rhizome of ammatory , anticanammatory ,9.In particular, electrospinning (ES) has emerged as a versatile technique for preparing nano and microfibers of natural and synthetic polymers. The electrospun fibers show fitness in diversified technological fields such as filtration, protective clothing, wound healing, and other biomedical applications ,11,12,13The core–shell methodology might contribute to overcoming some challenges associated with electrospinning techniques  such as the incorporation of high compound loading, preservation of the activity and presence of burst effect. The use of core–shell structures allows for better tuning of the release kinetics by tailoring the thickness of shell polymer, which acts as a diffusion barrier for the compound loaded in the core as well as a protection of the active molecule from the sunlight or from microbiological attack for example. Then, coaxial fibers allow for the bioactive molecule to be located either in the core or shell layer in a simple one-step process and to modulate the thickness of fibrous material triggering the drug release kinetic . MoreovePseudomonas aeruginosa PAO1, and Streptococcus mutans, two opportunistic pathogenic bacteria frequently associated with the infection of biomedical devices both in oral and in orthopedic implantology [Streptococcus mutans, moreover, is a Gram-positive bacterium present in the oral cavity and is one of the major contributors to dental biofilms [For these reasons, recently electrospinning technique was used in the treatment and prevention of biofilm-related infections, where the possibility to finely trigger the drug release may have a different impact on the bacterial/biofilm growth ,40,41. Bantology ,47,48. Tantology ,49,50. Sbiofilms ,52.S. mutans biofilm growth and activity. The high biocompatibility and the ability to control the oxidative stress of damaged tissue, make the synthesized membranes useful as scaffolds in tissue engineering regeneration, helping to accelerate the healing process and to prevent biofilm-associated infections.The results reported herein demonstrate the ability of the single-needle-spinned PCL-Cur and the coaxial membrane PCL-Cur/PLA (PCL in the core and PLA in the shell) to inhibit both PAO1 and Electrospinning conditions were optimized to produce fibrous mats with bead-less fibers ,54. By aAs shown in It is evident that the core containing the drug is thicker than the shell of PLA; the PCL core is 286 nm, whereas the PLA shell is 60.5 nm.Thermogravimetric analysis was performed for investigating the degradation temperature of the coaxial fibers compared to the pure polymers. The thermal decomposition up to 700 °C in the air of the electrospun fibers, either PCL-Cur or coaxial PCL-Cur/PLA, are shown in The degradation behavior of the coaxial membrane is higher than the PCL-Cur. This is due to the presence of PLA that shows lower thermal stability. The mechanical properties of PCL-Cur membranes were evaluated from stress–strain curves and compared to the coaxial system. The mechanical parameters are reported in Interestingly, the coaxial fibers show a higher mechanical modulus due to the strengthening of one material inside the other. At variance, the coaxial systems displayed a lower extensibility than PCL system as well as reduced stress at the breakpoint. This can be due to the dishomogeneity of the systems. However, the decrease of these parameters is not dramatic, and does not compromise the mechanical properties, also considering the increase of the elastic modulus.v/v), a medium that facilitates the solubilization of the curcumin. As shown in p < 0.001) more sustained curcumin release, compared to PCL-Cur, after 2 h of incubation and throughout the experimental period.As curcumin is poorly soluble in aqueous solutions, the release kinetic was determined in PBS/EtOH .Moreover, the coaxial membrane PCL-Cur/PLA showed a higher curcumin loading efficiency in comparison to the PCL-Cur membrane, incorporating 70 ± 3% and 56 ± 2% of curcumin, respectively. The obtained data are in accordance with the literature, confirming the capacity of the coaxial spinning to load the drug and control the initial burst release more efficiently with respect to single-needle spinning ,56. FurtBiofilm inhibition was analyzed at different times, as reported in p < 0.001) in both PAO1 and S. mutans biofilm formation only for the PCL-Cur and PCL-Cur/PLA, according to the release study.The results indicated a significant decrease -l-homoserine lactone (3-O-C12-HSL) and N-butyryl-l-homoserine lactone (C4-HSL) that promote the transcription of virulence genes  [rhlAB operon [S. mutans regulated the production of virulence factors via a two-component signal transduction system (TCSTS), consisting of a membrane-bound histidine kinase (HK) sensor protein, and a cognate cytoplasmic response regulator (RR) protein [S. mutans, the TCSTS consist principally in the operon ComAB/ComCDE where comC gene encodes competence-stimulating peptide (CSP), comD encodes HK sensor protein (ComD), and comE encodes an RR protein (ComE) [In general, QS is regulated by different autoinducer molecules in Gram-negative and Gram-positive bacteria . Indeed,olipids) ,59. Amonolipids) , are synB operon . Convers protein . In S. mn (ComE) .p < 0.001) decreased the mRNA levels of all genes tested, compared to control (gene expression level in biofilm formed in the presence of PCL and PLA membranes). In particular, for PAO1, the relative expression levels of the virulence genes rhlA, and rhlB were significantly decreased by 0.85-, and 0.83-fold, respectively, after 6 h of incubation in the presence of PCL-Cur, while by 0.72- and 0.73-fold after 6 h of incubation in the presence of PCL-Cur/PLA. This reduction was more evident after 24 h of incubation, reaching a downregulation of 0.67- and 0.53-fold for PCL-Cur and 0.53- and 49-fold for PCL-Cur/PLA. The same behavior was observed for S. mutans biofilm, where the treatment with PCL-Cur and PCL-Cur/PLA for 6 h repressed the relative expression levels of comC and comD to 77% and 72% (PCL-Cur), and 37% and 49% (PCL-Cur/PLA), respectively, as compared to control values. After 24 h of incubation, only the relative expression level of com D significantly (p < 0.001) was downregulated, reaching a 29% decrease compared to control for PCL-CUR/PLA.As shown in Pseudomonas aeruginosa PAO1 and the Streptococcus mutans growth curves.Antimicrobial activity was then analyzed at 6 and 24 h in the presence of prepared membranes. As reported in The reported results demonstrated that the curcumin released from electrospun membranes is able to affect biofilm formation without interfering with bacterial growth.2 group of the β-diketone moiety [Inflammation represents an adaptive physiological response to external and/or internal deleterious circumstances, including infection and tissue injuries. Extensive studies have suggested that continuous oxidative stress activates the inflammatory signaling cascade producing new free radicals that, in turn, lead to further oxidative stress, thus creating a cycle. This altered status can cause various chronic diseases such as cancer, atherosclerosis, Alzheimer’s disease, metabolic disorders, and so on ,65. Amone moiety ,70. Indee moiety .The ability of curcumin released from membranes to produces an antioxidant effect was evaluated by the DPPH radical scavenging assay, a simple and highly sensitive method to evaluate the free radical scavenging activity of antioxidants .As reported in To investigate the effect of curcumin extract on viability, human dermal fibroblasts (HDFs) were chosen as in vitro cell models. The results depicted in Taken together, these results demonstrate that the fabricated membranes could be employed as scaffolds in tissue engineering regeneration to control the oxidative stress of damaged tissue, helping to accelerates the healing process.l-lactide-co-d,l-lactide)  was purchased from NatureWorks . Tetrahydrofuran (THF pure-CAS: 109-99-9), Ethanol (EtOH purity > 96%-CAS 64-17-5) and Phosphate Buffer Solution (PBS-pH = 7 ± 0.02-CAS: 7558-79-4) were purchased from Carlo Erba Reagents (Cornaredo-Milano). N,N-Dimethylformamide (DMF-CAS 68-12-2) and Curcumin (Cur) were purchased from Sigma Aldrich . Cell Counting Kit-8 (CCK-8) assay and Lactate dehydrogenase assay were from Roche Applied Science . Human dermal fibroblasts were purchased from the American Type Culture Collection ATCC  and cultured in accordance with the manufacturer’s instructions. Fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), sodium pyruvate, L-glutamine, penicillin, and streptomycin were purchased from Hyclone .Poly(ε-caprolactone)  was purchased from Sigma Aldrich while poly (v/v) at 12% w/w. Curcumin was added to PCL solution at the drug to polymer ratio of 0.1:9.9 (w/w) and mixed for 4 h at 40 °C using a temperature-controlled stirring plate (300 rpm) to obtain a homogenous solution. Coaxial electrospun membranes were obtained by using a coaxial nozzle (EM-CAX-Ime electrospinning). Two separate volumetric pumps were used to process the polymeric solutions, prepared as previously described. The curcumin-loaded coaxial nanofibrous mats were processed by coaxial electrospinning; the drug-loaded solution constitutes the inner core, while the no-loaded solution is the outer shell. Before performing the experiment, each solution was fed in a 5 mL syringe pump. The sets of electrospinning conditions were reported in The electrospinning membranes were prepared by dissolving PCL and PLA in a solvent mixture THF/DMF  was used to produce fibrous membranes. The vertical setup was chosen to carry out the experiments. Core–shell nanofibers were obtained through a coaxial apparatus containing two concentric needles. The diameters of the inner and outer needles were 0.8 mm and 1.2 mm, respectively. The inner needle was 0.20 mm longer than the outer needle. For all the experiments, an aluminum collector was used to recover the electrospun nanofibers.SEM was carried out using a Quanta 200 F microscope  in high-vacuum mode. Before the analysis, electrospun membranes were covered with a thin film of gold using an Agar Automatic Sputter Coater  at 40 mA for 120 s prior to the analysis.TEM was performed on a FEI Tecnai 200 kV electron microscope  operating at 100 keV. The samples for the TEM observation were prepared by directly depositing the as-spun fibers onto the copper grids.TGA were carried out in an air atmosphere with a Mettler TC-10 thermobalance  from 25 °C to 800 °C at a heating rate of 10 °C/min.Mechanical properties were evaluated, in tensile mode, at room temperature using a dynamometric apparatus INSTRON 4301 . Experiments were conducted at room temperature with a deformation rate of 5 mm/min. Elastic modulus was evaluated in the deformation range of 0.1%. Data were averaged on five samples.2) were weighted and dissolved in methanol (1:5 w/v) for 60 min and curcumin concentration was measured as reported above by HPLC. The curcumin loading efficiency (LE) was calculated as percentage respect to the loaded drug as follows:Square pieces of membranes (ca. 2 cm2) were weighed and placed into individual amber vials. Considering the very low solubility of the curcumin in aqueous solutions, the release kinetic was performed at 37 °C in PBS/EtOH (90:10 v/v). At predetermined time intervals , supernatants were withdrawn, and the same amount of fresh solution was added back to the release medium. The curcumin concentration was measured using HPLC-UV with a linear elution gradient consisting of mobile phase A (0.1% acetic acid), B (Acetonitrile), and C (Methanol). The detection wavelength was set at 420 nm and Curcumin quantitation was based on a standard curve in PBS/EtOH (90:10 v/v). System control and data acquisition were performed using ChemStation software (Agilent Technologies). The results were presented in terms of cumulative release (percentage with respect to the loaded drug) as a function of time.The release of the curcumin from the membranes was determined as early described with some modifications . Brieflyv/v) for 0.5, 1, 8, 24 and 48 h and then ultrasonicated for 15 min. The dilution of the solutions was done in PBS/EtOH (90:10 v/v). Aliquots (500 μL) of those solutions were added to 2 mL of DPPH methanolic solution (60 μM) and kept in the dark at 37 °C for 1 h. The absorbance of the samples was determined at 517 nm using a microplate reader . A methanolic solution of free curcumin and polymers was used as a control. The percentage of inhibition of DPPH was calculated as follows:1 was the absorbance of the control (DPPH solution without sample) at 517; A0 was the absorbance at 517 of the sample at different concentrations with DPPH. The antioxidant activity was expressed as % with respect to free curcumin.Antioxidant activity, as the free radical-scavenging ability of curcumin-loaded nanofibers, was examined using 1,1-dipheny-l-2-picryl hydrazyl (DPPH) assay as reported by Amrati et al. with slight modifications . Samplesv/v % serum containing DMEM medium for 24 h at 37 °C to produce the sample extraction. Human dermal fibroblasts (HDFs) were cultured in 96-well tissue-culture polystyrene plate (TCPS) at 2 × 103 cells/well in serum-containing DMEM for 16 h to allow cell attachment. After that, the medium was replaced with an extraction medium and HDFs were incubated for a further 12, 24 and 48 h. At the end of the incubation period, all CCK-8 solutions were added to each well, and the plate was then incubated under cell culture conditions for 1–4 h. The optical density of formazan salt at 450 nm was measured using a Citation 3 Cell Imaging Multi-Mode microplate reader . The cytocompatibility of the membranes was expressed as a percentage relative to the control and calculated as:Indirect cytotoxicity evaluation of curcumin-loaded membranes was conducted according to ISO 10993-5 standard test method as reported by Conte et al. with slight modifications . SamplesLactate dehydrogenase (LDH) release measurements were based on the measurement of lactate LDH released into the growth media when the integrity of the cell membrane is lost. For this assay, HDF cells were treated with extracts in the same way as described above. At the end of the incubation time, 100 μL of the culture supernatants were collected to a well, and LDH activity was detected at 450 nm as reported by manufacturing protocol. As a positive control, cells were completely lysed with Triton X-100, according to Calarco et al. .Pseudomonas aeruginosa PAO1 (ATCC® BAA-47™) and Streptococcus mutans (ATCC® 25175) were purchased from ATCC, and cultured following the ATCC’s guidelines.7 CFU/mL and incubated at 37 °C and 200 rpm in a microplate reader (Cytation 3). As a control, the growth curve was obtained in the presence of PCL mat. At scheduled times (6 h or 24 h), the optical density (OD) at 600 nm was recorded.The capability of the curcumin-loaded membranes to inhibit bacterial growth was determined by monitoring the optical density (OD) at 600 nm of bacterial suspensions cultured in the presence of both PCL-Cur and coaxial PCL-Cur/PLA. All mats were cut of similar dimension, sterilized by UV radiation for 15 min at each side, and finally placed in a 12-well plate in the presence of 500 µL liquid broth as previously described . Briefly7 CFU/mL of S. mutans or PAO1 were added and the cultures were incubated statically at 37 °C in a humid atmosphere. PLA or PCL mats incubated in liquid medium broth were used as a negative control, while 750 µL of PAO1 (1 × 107 CFU/mL) or S. mutans (1 × 107 CFU/mL) were used as positive controls.To investigate the ability of the curcumin-loaded membranes to inhibit the biofilm formation, a similar amount of PCL-Cur, and PCL-Cur/PLA were sterilized by UV radiation for 15 min at each side, placed in a 48-well polystyrene plate, and biofilm developed as described by Di Salle et al. with some modifications . Brieflyw/v Crystal Violet was added to each well. After 30 min, excess solution was removed, and any extra stain was removed by washing with PBS. The stained biofilms were solubilized in 96% ethanol and quantified by measuring the optical density (OD) at 570 nm using a microplate reader . Measurements were carried out in triplicate for each membrane.The surface-adhered biofilm was quantified after 6, 12, and 24 h by the Crystal Violet (CV) assay. Each well was washed gently with sterile phosphate-buffered saline (PBS), and air-dried for 30 min. Then, a solution of 0.1% rhlAB genes for PAO1 and of comCD genes for S. mutans was quantified by real-time PCR (qRT-PCR). PAO1 and S. mutans biofilms were developed in the presence of synthesized membranes for 6 or 24 h, as previously described in a 48-well polystyrene plate. The bacterial pellet was then collected by centrifugation at 13,000× g for 10 min, and total RNA was extracted using TRIzol reagent  as previously described [To determine the ability of the membranes to interfere with the quorum-sensing mechanism underlying the biofilm maturation process, the mRNA level of escribed . Briefly16SrRNA), and the results expressed as mean ±SD. The comparative cycle threshold (2−ΔΔCt) method was used to determine the relative quantification.qPCR and data collection were performed on 7900HT Fast Real-time PCR System . The reactions were performed according to the manufacturer’s instructions using SYBR Green PCR Master Mix . All reactions were run in triplicate, normalized to the housekeeping gene  with Tukey’s post-hoc test were used for statistical comparison. The difference was regarded as statistically significant when p < 0.05. All the data were analyzed with the GraphPad Prism version 6.01 statistical software package .Results were expressed as mean ±  standard deviation (SD). Student’s Pseudomonas aeruginosa PAO1 and Streptococcus mutans—two opportunistic bacteria frequently associated with human infections. In particular, we observed a more important effect, especially after 12 h of incubation, with the coaxial respect to the single-needle electrospun membrane, showing a reduction of 23 ± 5%, 33 ± 4% in PAO1 and S. mutans biofilm formation, respectively. Our data also demonstrated the ability of CUR-loading membranes to inhibit signal-based biofilm formation, lowering the expression of several genes involved in the quorum-sensing mechanism that led to biofilm maturation.In this study, electrospinning was used to produce nanofibrous membranes with the ability to release curcumin in a continuous and sustainable mode, eliciting both antibacterial and antibiofilm activity on Moreover, the presented results demonstrated good biocompatibility of both PCL-Cur, and PCL-Cur/PLA and a good antioxidant capacity already after 30 min of incubation.Taken together, these results demonstrate that the fabricated membranes could be employed as scaffolds in tissue engineering regeneration to control the oxidative stress of damaged tissue, thus helping to accelerates the healing process.


Psoriasis is a chronic skin disease affecting 2–3% of the global population. The proinflammatory IL-17A is a key cytokine in psoriasis. Accumulating evidence has revealed that IL-36γ plays also a pathogenic role. To understand more precisely the role of the IL-17A–IL-36γ cytokine network in skin pathology, we used an ear injection model. We injected IL-17A or IL-36γ alone and in combination into the ear pinnae of mice. This resulted in a significant increase in ear thickness measured over time. Histological evaluation of IL-17A + IL-36γ-treated skin showed a strong acanthosis, hyperparakeratosis and infiltration of neutrophils. The same histological features were found in mice after injection of IL-36γ alone, but to a lesser extent. IL-17A alone was not able to induce psoriasis-like changes. Genes encoding proteins of the S100 family, antimicrobial peptides and chemo-attractants for neutrophils were upregulated in the IL-17A + IL-36γ group. A much weaker expression was seen after the injection of each cytokine alone. These results strengthen the hypothesis that IL-17A and IL-36γ drive psoriatic inflammation via a synergistic interaction. Our established intradermal ear injection model can be utilized in the future to monitor effects of various inhibitors of this cytokine network. IL36A, B and C in humans and Il1F6, 8 and 9 in mice, belong to the IL-1 cytokine family and possess multiple roles in host immunity and several inflammatory diseases [Psoriasis is a chronic skin disease caused by the excessive secretion of inflammatory cytokines affecting 2–3% of the global population . Psoriatdiseases ,21. Amondiseases ,19. So fdiseases . The roldiseases , and whidiseases . It is hdiseases . It was diseases . Additiodiseases . Furtherdiseases . A phasediseases . Generaldiseases . Il-36 cdiseases . To furtEight-week-old female C57BL/6 mice were used. They were purchased from Janvier . All animal procedures and experiments were conducted in accordance with the German federal law regarding the protection of animals. Protocols were approved by the administration of the “Landesamt für Umwelt, Natur und Verbraucherschutz” . Each group consisted of 5 animals.The following recombinant murine cytokines, diluted in PBS, were used: IL-17A  and IL-36γ . The cytokines IL-17A and IL-36γ were carefully injected intradermally, either separately or combined, into the ear pinnae  by using a Hamilton Syringe (33 gauge). The control group received injections with PBS. Accordingly, four groups  were included in the study.Ear thickness measurement was performed daily with a pocket thickness gauge . The measuring tool was positioned at the ear pinnae of the mice.After four days of consecutive treatment, the mice were euthanized, and both ears were collected. One part of the ear was fixed in formalin and embedded in paraffin, followed by haematoxylin and eosin (H&E) staining. Moreover, cryosections of the ears were stained for neutrophils with a Ly-6G antibody .Examination and photographic documentation of the H&E-stained paraffin-embedded sections of formalin-fixed ear tissue and the cryosections were performed using a DMIL microscope .One part of the ear was harvested for RNA isolation. Quantitative RT-PCR experiments were performed directly afterwards. According to the manufacturer’s instructions, the Nucleo Spin RNA kit  was used for total RNA extraction. Residual genomic DNA was removed by rDNase (RNase-free) digestion. The tissues of the mouse ears were mechanically disrupted and homogenized by using a tissue lyser . A total of 1 µg of RNA was reverse transcribed into cDNA by utilizing High Capacity RNA-to-cDNA Master Mix . TaqMan experiments were carried out on an ABI PRISM 7300 sequence detection system  using Assay-on-Demand gene expression products  for Defb4 (Mm00731768_m1) and S100a8 (Mm00496696_g1). An Assay-on-Demand product for Hprt mRNA (Mm00446968) was used as an internal reference to normalize the target transcripts.We have randomly selected 3 out of 5 initially generated samples and subjected them to an RNA-Sequencing experiment, consisting of library preparation, sequencing, and subsequent analysis.Quality checks of RNA samples were performed with the TapeStation 4200 using the Agilent RNA ScreenTape Assay kit. Quantification was performed using the Quantus RNA System . cDNA libraries were produced employing the TrueSeq Stranded Total RNA Library Preparation Kit with Ribo-Zero Gold Kit according to the manufacturer’s instructions , using 500 ng input RNA or 10 µL for the immunoprecipitated samples, respectively. The cDNA libraries’ quality and quantity were assessed using the 4200 TapeStation (D1000 screen tape assay) and the Quantus dsDNA system, respectively.The libraries were run on an Illumina NextSeq 500 platform using the High Output 150 cycles kit . The run includes the primary analysis workflow, mainly consisting of template generation, imaging, and base calling using RTA 2.0 onsite. Then, the run resulted in an average sample size of 88.45 million reads and a sample size standard deviation of 21.23 million reads. The samples were sequenced on a NextSeq 500 sequencing system with the following run parameters: 76 cycles for read 1, 76 cycles for read 2, and 6 cycles for index 1. The cDNA libraries were generated using a TruSeq Stranded Total RNA Sample Preparation Kit .https://support.illumina.com/sequencing/sequencing_software/bcl2fastq-conversion-software.html (accessed on 14 January 2020)). Quality control checks of the RNA-Seq data were performed with fastqc, available online (http://www.bioinformatics.babraham.ac.uk/projects/fastqc (accessed on 14 January 2020)). Subsequently, the reads were aligned to the mm10 reference genome sequence using STAR (v2.5.2b) with default parameters [http://subread.sourceforge.net (accessed on 14 January 2020)) [The RNA-Seq primary analysis was performed with an in-house pipeline embedded in the QuickNGS workflow management system : the genrameters . In the y 2020)) .p-value < 0.05). p-values were corrected for multiple testing with the p.adjust function from the R package stats (v 4.0.3) using the Benjamini Hochberg correction.Differential expression analysis was performed in R using the DESeq2 package (v1.30.0) : first, p-value of at most 0.05.Gene Set Enrichment Analysis was performed with the piano package (v 2.6.0) using the “runGSA” method . For stap-value < 0.01 considered as highly significant. Gene counts were normalized gene-wise. Each measured expression value of a gene was divided by the sum of this gene’s expression values across all samples and upscaled by a scaling factor of 106. Afterward, each normalized value was centered around the average expression of this gene across all samples. Finally, the counts were rescaled by the thousandfold of the maximal normalized count. Based on this, we constructed the heatmap matrix of the selected GO-terms and generated the heatmap with the R-package pheatmap (v1.0.12).We have selected from each selected gene set all upregulated genes with high significance in the combined injection compared to PBS injection, with genes with a fold change ≥2 and a The heatmap of the top 50 variable genes was generated using the same normalization, scaling, and centering procedure described for the heatmap of GO-terms. The top 50 genes were selected based on their variability across all samples. We have used the variance as a measure of variability.p < 0.05, ** p < 0.01 and *** p < 0.001 were considered significant and are indicated in the figures.Data are given as arithmetical means ± standard deviation (SD). Mann–Whitney U test and Bonferroni multiple comparisons tests were performed with GraphPad PRISM version 7 . Values of * This study aimed to establish an IL-17A and IL-36γ intradermal ear injection model of psoriasis to elucidate the role of both cytokines in the complex pathogenesis of the disease. While former studies used IL-23 to induce psoriasis in mouse models ,38, we wWhile the increase in ear thickness is an indicator of vascular permeability and oedema, an increased epidermal thickness could be attributed to differences in the number of actively proliferating cells within the epidermal layers of the groups. The ear and epidermal thickness results were concordant with visually apparent skin changes D. The moNext, we performed gene expression profiling using RNA-Seq. Nineteen gene sets that play a pivotal role in the psoriasis pathomechanism were selected A. While We performed principal components analysis (PCA) on the expression profiles of the 12 samples, then we selected the first two principal components (describing ~90% of the variance in the data). The corresponding PCA plot showed that replicates of the same treatment group were generally clustering. All groups were well separated along PC1, contributing 78% to the data variance. We concluded that PC1 explains the effect of the treatment, with the effect increasing from left to right. In contrast, we supposed that differences along PC2 were mainly due to technical effects as well as natural variability A.The similarities of the differentially treated groups were further confirmed by the Similarity Heat Map B. The reAs mentioned before, neutrophils and T-cells play a crucial role in developing and maintaining psoriasis inflammation. We expected that in the RNA-seq dataset, obtained from biopsies of mouse ears that include infiltrating cells, signatures of both neutrophils and T cells should be detectable. Therefore, genes that are preferentially expressed either in neutrophils or in T cells were evaluated ,40. AfteThe WHO classifies psoriasis as one of the most serious non-communicable diseases. It is characterized by aberrant and enhanced proliferation of epidermal keratinocytes driven by immune mediators including IL-23, IL-17 and IL-36 . PreviouWe demonstrated that IL-36γ and IL-17A individually led to a significant increase in ear and epidermal thickness, which was further increased by the combined administration of both cytokines. This strong increase in ear thickness is also observed in IL-23-induced mouse models of psoriasis described in the literature, concordant with our results . InjectiCxcl9 and Cxcl10 were significantly upregulated, which are both important chemo-attractants for neutrophils. It has been reported that intraperitoneal treatment with a neutrophil-depleting antibody (anti-Ly-6G) caused a dramatic reduction in epidermal thickness in a psoriasis mouse model and suggested that blocking neutrophil function may have therapeutic benefit in inflammatory human skin disorders [The role of neutrophils in psoriasis has been known for a long time. Neutrophil chemotaxis is significantly upregulated in psoriasis patients . The infisorders . Therapiisorders ,46.The role of TH1 in the disease is well known. A feedback loop has been postulated in which TH1 activation leads to the release of IFNγ and TNFα, which in turn stimulate the secretion of Cxcl10 by keratinocytes, lymphocytes, and other immune cells . The relGenes involved in angiogenesis and blood vessel development pathways, which are associated with psoriasis, are significantly upregulated by the combination of IL-17A + IL-36γ, and to a lesser extent in response to IL-36γ alone. Our results are in line with findings that highlighted the role of IL-36γ in vascular activation, and the induction of angiogenesis in a VEGF-A-dependent manner in the context of psoriatic lesions . AdditioIl1b, whose proinflammatory role in psoriasis has been described previously [The stimulation with IL-17A + IL-36γ upregulated numerous psoriasis-associated genes. This included, for example, the RIKEN cDNA 2610528A11 gene, which is a marker for psoriasis in humans. Moreover, the mRNA for 2610528A11RIK (Gpr15l) was significantly decreased upon treatment with the antibody AIN457 (Secukinumab), which was simultaneously accompanied by a marked improvement in clinical findings . Furthereviously ,52.The upregulation of genes encoding proteins of the S100 family is in line with previous studies, which showed that this protein family plays a crucial role in psoriasis, especially in inflammation, angiogenesis, and keratinocyte proliferation . S100A8/Krt16 was demonstrated in a three-dimensional psoriasis mouse model [Krt16 was upregulated in response to IL-36γ, either alone or in combination with IL-17A.Furthermore, a significant upregulation of genes belonging to the Krt family involved in keratinization, and keratinocyte differentiation was found. Comparable to our study, an upregulation of se model . HoweverLce3a and Lce3b, which are expressed only in the epidermis and in oral epithelia. The encoded proteins are not only relevant in the late cornified envelope, but also possess antimicrobial activity [IL-17A and IL-36γ induced the expression of activity . In lesiactivity .Defb3 and Defb4 were upregulated by IL-36γ and synergistically by co-stimulation with IL-17A + IL-36γ. Interestingly no upregulation was detected after stimulation with IL-17A alone. Our results differ from a previous study [us study , in whicIn conclusion, intradermal injection of IL-17A + IL-36γ into the ear pinnae of mice provides an in vivo model to investigate psoriasis, which mimics both histological and gene expression aspects of the disease. Our results strengthen the hypothesis that IL-17A and IL-36γ drive psoriatic inflammation via a synergistic interaction. In severe cases of psoriasis, the combined targeting of IL-36 and the IL-23–IL-17 axis may be a new beneficial therapeutic approach. Monoclonal IL-36 receptor-blocking antibodies such as Spesolimab have already proven their efficacy in generalized pustular psoriasis in Phase II clinical trials. Our established intradermal ear injection model can be utilized in the future to monitor the effects of various inhibitors, e.g., biologicals, and topically applied small molecules, that target the IL-17A and IL-36γ cytokine network.


Accumulating evidence suggests that oxidative stress is involved in the development of chronic obstructive pulmonary disease (COPD) and its progression. Activity of extracellular superoxide dismutase (ecSOD), the only extracellular enzyme eliminating superoxide radicals, has been reported to decline in acute exacerbations of COPD (AECOPD). However, the association between serum ecSOD activity and 1-year all-cause mortality in AECOPD patients remains unclear. The objective of our study was to explore the usefulness of ecSOD activity on admission in AECOPD as an objective predictor for 1-year all-cause mortality.We measured serum ecSOD activity in AECOPD patients on admission in a prospective cohort study. We also recorded their laboratory and clinical data. Multivariate Cox regression was used to analyze the association between ecSOD activity and the risk of 1-year all-cause mortality. Restricted cubic spline curves were used to visualize the relationship between ecSOD activity and the hazard ratio of 1-year all-cause mortality.p = 0.004) and had increased levels of tobacco consumption . Having an ecSOD activity ≤ 98.8 U/ml was an independent risk factor of 1-year all-cause mortality after adjustment for baseline differences, clinical variables and comorbidities .A total of 367 patients were followed up for 1 year, and 29 patients died during a 1-year follow-up period. Compared with survivors, the non-survivors were older (79.52 ± 8.39 vs. 74.38 ± 9.34 years old, Lower serum ecSOD activity was a strong and independent predictor of 1-year all-cause mortality in AECOPD patients. Chronic obstructive pulmonary disease (COPD), a systemic inflammatory disease, is the third leading cause of mortality worldwide . ExacerbSOD3 have been associated with declining lung function in COPD  is the only extracellular scavenger of the superoxide radical and has been reported to be markedly higher in the lung . EcSOD p in COPD  and that in COPD . Thus, eIn COPD patients suffering from exacerbations, it was found that the antioxidant pathway was downregulated, and serum ecSOD activity was decreased , 16. Inc1)/forced vital capacity (FVC) ratio of <0.7] and AECOPD  at admission, defined according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria  from January 01, 2016–January 10, 2020. Participants were diagnosed with COPD  , and other analyses were performed using SPSS software, version 16 .The primary outcome was 1-year all-cause mortality. Parametric data were expressed as the mean ± standard deviation. Categorical variables were expressed as percentages. We compared groups using the independent samples  package . The ent(PaCO2)] –24. The (PaCO2)] . All anap = 0.004) and had more COPD patients with GOLD stage four (24.1 vs. 16.6%) than did the survivor group. Compared with the survivor group, the non-survivor group also had more smokers (82.7 vs. 77.2%) and increased tobacco consumption .A total of 396 consecutive patients were recruited and after screening for inclusion and exclusion criteria, 382 patients were enrolled, and ecSOD activity data for 11 of these patients were unavailable. Four patients were lost to follow-up after discharge, and 367 patients completed a 1-year follow-up and were included in the study analysis . The meap < 0.001). We separated the entire cohort into two groups according to the optimal cut-off value determined by maximally selected log-rank statistics to analyze differences in prognostic consequences  died in the entire cohort during follow-up. We first explored the association between 1-year all-cause mortality and ecSOD activity as a continuous variable with the use of Cox regression analysis. We found that ecSOD activity was negatively correlated with the risk of 1-year all-cause mortality [HR, 0.95; 95% confidence interval (CI): 0.93–0.97; equences . The optp < 0.001). The survival rate in the ecSOD activity ≤ 98.8 U/ml group was lower than that in the ecSOD activity > 98.8 U/ml group . In subgroup analysis, there were significant differences in survival curves between the two groups for both never smokers . After adjusting for baseline characteristics (model 2), the HR was 4.11 . After further adjustments for clinical variables and comorbidities (model 3), the relationship between the ecSOD activity ≤ 98.8 U/ml group and 1-year all-cause mortality remained significant . The restricted cubic spline curve (p for non-linearity < 0.001).We constructed three models to clarify the relationship between the ecSOD activity and 1-year all-cause mortality with the use of multivariate Cox proportional hazards analysis . The unane curve , based op = 0.145) or smoking status  during exacerbations were not significantly different. For current smoker, the ecSOD activity in the non-survivor group and the survivor group were 111.80 ± 26.92 U/ml and 113.94 ± 18.95 U/ml (p = 0.812), respectively. For former smoker, the ecSOD activity in the non-survivor group and the survivor group were 92.49 ± 20.33 U/ml and 111.44 ± 19.33 U/ml (p <0.001), respectively. For never smoker, the ecSOD activity in the non-survivor group and the survivor group were 84.10 ± 28.65 U/ml and 113.91 ± 15.88 U/ml (p < 0.001), respectively.We investigated the association between serum ecSOD activity and 1-year all-cause mortality in our prospective cohort study of AECOPD. We found that lower serum ecSOD activity in AECOPD patients might be a significant predictor for their 1-year all-cause mortality. To our knowledge, this is the first study to highlight the relationship between ecSOD activity and 1-year all-cause mortality in AECOPD. Exacerbations of COPD were associated with airway and systemic oxidative stress-induced inflammation . EcSOD hSOD3 R213G heterozygotes (mutation causes elevated serum ecSOD levels) had a decreased risk of hospitalization or mortality from COPD. Furthermore, Singh et al. (p < 0.001). These studies provided evidence that serum ecSOD activity had potential as a prognostic factor in AECOPD patients.The restricted cubic spline curve  visualizh et al.  proposedh et al. , 32. Con1, were associated with decreased ecSOD activity . The influence of patients taking vitamins and other supplements daily on ecSOD activity was a long-term effect. We considered that the ecSOD activity was an antioxidative indicator that reflected the body's comprehensive antioxidant capacity. The effect of taking vitamins could be reflected by ecSOD activity. Our study aimed to explore the relationship between ecSOD activity during exacerbations and 1-year all-cause mortality, and lower SOD activity represented more severe oxidative stress in patients within exacerbations. Therefore, we did not exclude patients taking vitamins and other supplements. Fourth, we could not be certain whether serum ecSOD activity reflected those in the lungs. There is no direct evidence to show the relationship between serum ecSOD activity and lung ecSOD activity in COPD. However, asthma studies have shown that airways as well as blood monocytes and neutrophils had lower MnSOD activity in asthma patients than in control subjects , the Natural Science Foundation of Guangdong Province (2017A030313887), the Guangzhou Science and Technology Project (201707010232), and the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01S155).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Furthermore, in a rat model of SCI, PA effectively improved neurological deficits. Overall, these results prove that PA exerts neuroprotective effects by maintaining BSCB integrity and thus be a promising candidate for SCI treatment.Spinal cord injury (SCI) is a destructive and complex disorder of the central nervous system (CNS) for which there is no clinical treatment. Blood-spinal cord barrier (BSCB) rupture is a critical event in SCI that aggravates nerve injury. Therefore, maintaining the integrity of the BSCB may be a potential method to treat SCI. Here, we showed that patchouli alcohol (PA) exerts protective effects against SCI. We discovered that PA significantly prevented hyperpermeability of the BSCB by reducing the loss of tight junctions (TJs) and endothelial cells. PA also suppressed endoplasmic reticulum stress and apoptosis  Spinal cord injury (SCI) is a disorder associated with high mortality that not only leads to physical disabilities but also imposes a large economic burden . NeuroloThe pathophysiological process of secondary nerve injury is mostly related to blood-spinal cord barrier (BSCB) rupture . The BSCOxidative stress is important for BSCB disruption in various pathological conditions . Under sRecently, studies have also confirmed that mitochondrial damage results from ER stress and is coupled with ER stress-mediated apoptosis . MitochoPatchouli alcohol  is a natural tricyclic sesquiterpene isolated from patchouli that has traditionally been used to treat infectious diseases. Previous studies have shown that PA has anti-influenza , antitumPatchouli alcohol was provided by Chengdu Herbpurify Co., Ltd., and the purity was 98%. Endothelial cell medium (ECM) and fetal bovine serum (FBS) were provided by Invitrogen . Anti-Akt, anti-p-Akt (Ser473), anti-cleaved-caspase-3, anti-cleaved-caspase-9, anti-Bax, anti-Bcl-2, anti-CHOP, anti-glucose-regulated protein (GRP-78), anti-ATF-6, and anti-GAPDH antibodies were provided by Santa Cruz Biotechnology . HRP-conjugated goat anti-rabbit and anti-mouse IgG were provided by Cell Signaling Technology Inc. . An enhanced chemiluminescence (ECL) kit was provided by Bio-Rad . LY294002 (a PI3K/Akt inhibitor) and all other reagents were provided by Sigma.6 cells in 5 ml of medium were added to each Petri dish and subsequently placed in an incubator . The cells spread on the bottom of the plate after 24 h and reached confluence in 3–4 days. To simulate spinal cord injury in vitro, HBMECs were exposed to oxygen-glucose deprivation (OGD). The medium was changed to sugar-free basic medium. The cells were then cultured in 60-mm or 35-mm Petri dishes coated with poly-D-lysine and starved overnight in 0.5% FBS. Next, HBMECs were cultured in hypoxic chambers  for 12 h (oxygen concentration <0.2%). Subsequently, the cell medium was replaced with ECM, and PA  was added.Human brain microvascular endothelial cells (HBMECs) were obtained from a commercial source  and cultured in ECM. Approximately 1 × 10To determine whether PA can maintain the integrity of HBMECs, HBMECs were pretreated with natural compounds (10 μM for the drug screening assays) and varying concentrations of PA. Endothelial monolayer permeability was evaluated based on the intensity of FITC-dextran . HBMECs Sixty female Sprague-Dawley rats weighing between 200 and 220 g were obtained from the Animal Center of the Chinese Academy of Sciences  and bred in a specific pathogen-free (SPF) laboratory. After 1 week of adaptation, the rats were anesthetized with 2% (w/v) pentobarbital sodium . Rats in the sham group endured the exposure of spinal cords while other rats were subjected to SCI. Then a laminectomy was performed at the T9 vertebra. For SCI rats, when the spinal cord was fully exposed, a MASCIS Impactor with a 2.5-mm diameter tip and a 10-g rod with a drop height of 12.5 mm  was used to induce contusion SCI in rats. Post-operatively, carprofen (5 mg/kg s.c.) was used to relieve the pain of the rats and the rats were randomly divided into three groups . PA was suspended in 0.1% Tween 80 in phosphate-buffered saline (PBS) and administered intraperitoneally at a dosage of 10 mg/kg/day for consecutive 56 days. Rats from the sham and SCI groups received an equal volume of 0.1% Tween 80 instead. The rats were housed following an injury after the muscles and skin were closed in layers, and the bladder was emptied manually twice a day. All the animal experiments were conducted in accordance with the ethics committee of the National Institutes of Health, China, and performed according to the National Institutes of Health Guide for Care and Use of Laboratory Animals .Blood-spinal cord barrier maintenance was evaluated based on the extravasation of Evans blue dye . One dayThe Basso, Beattie, and Bresnahan (BBB) locomotor rating scale was used to assess the locomotor ability of the animals on a 22-point (0–21) scale assessing hindlimb locomotor function and open-field locomotion . Before 2 and initially perfused with 0.1 M phosphate-buffered saline (PBS) via cardiac puncture. For hematoxylin and eosin staining, immunohistochemistry, the spinal cords were removed, post-fixed in 4% paraformaldehyde overnight, and subsequently embedded in paraffin. The paraffin-embedded sections (5 μm thick) were mounted on slides for the subsequent step. For molecular studies, the spinal cord segment at the injury site was immediately isolated and frozen at −80°C.The animals were divided into three groups: sham, SCI, and SCI + PA. At specific time points after SCI, an equal number of rats was selected from each group, sacrificed by exposure to excessive COAfter the cells were grouped and processed, total RNA was extracted using TRIzol reagent and then reverse transcribed into cDNA. Next, the samples were denatured at 94°C for 10 min and amplified at 94°C for 15 s, 60°C for 60 s, and 72°C for 60 s for 40 cycles. The primer sequences were as follows: ZO-1 sense: 5′-AGGTGAAACTCTGCTGAGCC-3′; ZO-1 antisense: 5′-TCCAGTGTGGCAAG CGTAG −3′; Claudin-5 sense: 5′-GAACTACGTCTAAGGGCGGG-3′; Claudin-5 antisense: 5′-AATTCAGCGGTG GTCGTCAT −3′; GRP78 sense: 5′-GTGCCCACCAAGAAGTCTCA −3′; and GRP78 antisense: 5′-TTTTGTCAGG GGTCGTTCACC −3′. An RT-PCR system (ABI) was used for quantification, and all the reactions were performed three times.To measure the necrosis or death of motor neurons, paraffin-embedded spinal cord sections were stained with hematoxylin and eosin according to the manufacturer’s instructions. For immunofluorescence, when the cells were approximately 70% confluent, they were fixed with 4% paraformaldehyde for 15 min at room temperature. Next, the cells were washed three times in PBS for 5 min. For immunohistochemistry, spinal cord sections were incubated in citric acid buffer (pH 6.0) in a microwave for antigen retrieval and cultured at room temperature for 1 h in blocking buffer. The blocking buffer comprised 5% bovine serum albumin (BSA) in PBS-0.1% Triton X-100 (Sigma). The slides were incubated at 4°C overnight with the following primary antibodies: ZO-1 , β-Catenin , and Claudin5 . Following primary antibody incubation, the slides were incubated with secondary antibody, and the cells were then incubated with the nuclear marker Hoechst 33258 . All the images were captured using a confocal fluorescence microscope .Protein extraction reagents were used to extract protein from spinal cord tissues and HBMECs. All the proteins from the tissues and cells were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with the following primary antibodies overnight at 4°C for analysis: ZO-1 , β-Catenin , and Claudin5 . GAPDH  was used as a loading control. Alexa Fluor 568-, Alexa Fluor 488-, or Alexa Fluor 647-conjugated donkey anti-rabbit or anti-mouse secondary antibodies were used. The signals were visualized using a ChemiDocXRS + Imaging System (Bio-Rad) for statistical analysis. All the experiments were repeated three times.5 cells/well and allowed to proliferate for 48 h. Next, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 3 h in a 37°C incubator at 5% CO2. Finally, the plate was placed in a microplate reader, and the absorbance was measured at 450 nm. The experiment was repeated three times.Cell Counting Kit 8 (CCK-8) was used to measure the toxic effect of PA on HBMEC proliferation according to the manufacturer’s instructions. HBMECs were induced to proliferate and centrifuged to generate a cell suspension. The PA groups were treated with 1, 2.5, 5, 10, 20, 50, or 100 μmol/L PA, and the untreated group served as a control. Each group of cells was then seeded in 96-well plates at a density of 1 × 10in vivo. Images were taken using a Nikon ECLIPSE Ti microscope . The apoptosis ratio of HBMECs was determined using a PI/Annexin V-FITC kit (Invitrogen). Next, a FACScan flow cytometer  was employed for analysis according to the kit’s manual.The One-Step TUNEL Apoptosis Assay Kit  was used to evaluate DNA fragmentation Mitochondrial JC-1 fluorescence was measured to determine the mitochondrial membrane potential (MMP) . At low t-test or one-way analysis of variance (ANOVA) and Dunnett’s post hoc test were implemented for pairwise comparisons between all the experimental groups unless otherwise stated. P < 0.05 was considered statistically significant.All the data were expressed as means ± standard error of the mean (SEM). Either two-tailed Student’s in vitro, and OGD was used to mimic the microenvironment after SCI. The RT-PCR results are shown in ∗P < 0.05).Several drugs that may have therapeutic effects on OGD-exposed HBMECs were screened. The FITC-dextran assay showed that, compared with other drugs, PA had a more significant effect on decreasing the permeability of HBMECs in a dose-dependent manner . The CCK∗∗P < 0.01). The expression of claudin-5, β-catenin, and P120-catenin was significantly increased in the PA-treated group compared with that in the OGD-exposed group . We also performed immunofluorescence to more intuitively visualize the changes in protein expression. The images . The above results indicated that PA effectively reduces the loss of TJ and AJ proteins induced by OGD, thereby reducing the permeability of the BSCB.Tight junctions and AJs are vital parts of the BSCB according to previous reports . To confe images  showed t∗P < 0.05; ∗∗P < 0.01). ∗P < 0.05; ∗∗P < 0.01). PA treatment effectively inhibited the excessive activation of early ER stress, suggesting that PA treatment reduces the accumulation of UPs in the ER during ER dysfunction. PA treatment also effectively inhibited the expression of CHOP when the ER was overactivated by stress (∗P < 0.05). The immunofluorescence results showed that PA reduced the expression of CHOP induced by OGD (∗∗P < 0.01). The above results indicate that PA reduces the expression levels of ER stress-related proteins by alleviating the early UP response in the ER, reducing the protein expression level of CHOP and reducing the damage resulting from excessive ER activation.The essence of the ER stress response is the accumulation of UPs. In the early stage, molecular chaperones such as GRP78 are recruited to allow adaptation to UP accumulation. When decompensated, the transcription factor CHOP is activated to induce apoptosis. The expression of the CHOP gene is positively regulated by transcription factors such as activating transcription factor 6 (ATF6), XBP-1, and ATF4. PA treatment reduced the expression levels of the ER stress-related molecules GRP78, ATF6, and DDIT3 under OGD conditions . Additionally, PA effectively increased Bcl-2 expression and inhibited Bax and C-casp3 expression when the ER was overactivated by stress . Furthermore, we applied JC-1 to measure the MMP. Normally, JC-1 forms aggregates that emit strong red fluorescence in the mitochondrial matrix; however, in damaged mitochondria, JC-1 exists in the cytoplasm as a monomer because of the loss of membrane potential and produces green fluorescence. PA reduced OGD-induced mitochondrial death . Additionally, TUNEL staining was used to detect apoptosis in HBMECs . The results proved that PA inhibited endothelial cell apoptosis after OGD treatment. The above results suggested that PA inhibits the loss of mitochondrial function and reduces the expression levels of apoptosis-related proteins, thereby reducing endothelial cell apoptosis.Endoplasmic reticulum stress induces the death of mitochondria and leads to apoptosis . The Bcl∗∗P < 0.01; ∗P < 0.05). TG induced HBMEC apoptosis after OGD exposure and PA reversed this effect . Thus, ER stress induces mitochondrial damage and subsequent cell apoptosis, and PA prevents mitochondrial damage by inhibiting ER stress.To verify the relationship between ER stress and mitochondrial damage, the ER stress inducer thapsigargin (TG) was applied. TG increased the expression levels of GRP78, C-caspase3, and Bax and decreased β-catenin and claudin-5 expression, whereas PA treatment reversed the effects of TG . Furthers effect . AdditioTo investigate whether PA inhibits ER stress activation in endothelial cells in an AKT-dependent manner, HBMECs were treated with LY294002, a classic PI3K inhibitor. The phosphorylation of AKT was increased by PA treatment , and LY2∗P < 0.05; ∗∗P < 0.01). Western blot analysis also proved that PA treatment decreased the protein expression levels of Bax and C-casp3 . Immunofluorescence showed that PA increased connexin levels and restored the function of the BSCB (∗∗P < 0.01). Overall, our experimental results showed that, after SCI, the ER stress level increases, the expression of junction proteins is simultaneously reduced, and the expression of apoptosis-related proteins is also increased, causing destruction of the BSCB. PA treatment effectively reverses these consequences.To prove that the ER stress level is increased and BSCB is disrupted after SCI, a rat model of SCI was established. The rats were intraperitoneally injected with PA. After 1 day, tissues were removed for protein extraction. Western blot analysis proved that β-catenin and Claudin-5 levels were negatively correlated with the protein expression of GRP78 and that PA treatment effectively increased the protein expression levels of β-catenin and Claudin-5 (∗∗P < 0.01). Evans blue dye penetration in the PA treatment group was lower than that in the SCI alone group (∗P < 0.05). The above results suggested that PA treatment can improve the permeability of the BSCB in rats with SCI. The three groups of rats were subjected to behavioral tests 0, 3, 7, 14, 28, and 56 days after modeling. The BBB locomotor scale . The scores of the rats in the SCI group and those in the PA treatment group on the inclined plate test were different at 7 days. The average score and standard deviation were significantly higher in the PA treatment group than in the SCI group at 56 days. Thus, PA effectively improves the locomotor ability of rats with SCI.Evans blue dye extravasation was assessed to evaluate the impact of PA on the BSCB. Spinal cord tissues from the sham group showed almost no Evans blue penetration, and Evans blue dye extravasation in the SCI group was significantly higher than that in the PA treatment group at 1 and 3 days . The peror scale  and inclor scale  were perSpinal cord injury is often accompanied by excitotoxicity and oxidative damage followed by BSCB dysfunction, microvascular injury, and inflammation . The BSCAs a type of naturally tricyclic sesquiterpene, PA exerts protective effects in multiple physiological processes, such as the suppression of inflammation. Previous studies have shown that PA has neuroprotective activities . Howeverin vitro and in vivo. CHOP is proapoptotic in cell apoptosis and increases rapidly under ER stress  disorders accompanied by BSCB dysfunction. However, our study did not clearly elucidate some questions. We will focus our attention on the specific mechanisms in the future. Taken together, our results suggest that based on its effects and mechanisms, PA may contribute to SCI treatment.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The animal study was reviewed and approved by Wenzhou Medical University.CH, WZ, HJ, and QW designed the experiments. HQ, FQ, YW, and CD performed the vitro the experiments. JZ, MY, ZL, and HQ performed the vivo experiments. WZ, FC, and CH wrote the manuscript. XW, QW, HJ, and HQ revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The LNA employs a resistive shunt-feedback structure with a feedforward noise-canceling technique to accomplish both wideband impedance matching and low noise performance. A complementary CMOS topology was also developed to cancel out the second-order harmonic distortion and enhance the amplifier linearity. A high-frequency ultrasound (HFUS) and photoacoustic (PA) imaging front-end, including the proposed LNA and a variable gain amplifier (VGA), was designed and fabricated in a 180 nm CMOS process. At 80 MHz, the front-end achieves an input-referred noise density of 1.36 nV/sqrt (Hz), an input return loss (S With the recent advancements of high-frequency >30 MHz) ultrasound transducers , such as0 MHz ultIn HFUS and PA imaging, ultrasound transducers are utilized to detect the acoustic pressure transients and to generate electrical signals accordingly. The electrical signals are then processed by the ultrasonic imaging receiver, in which the low-noise amplifier (LNA) is the key component. To support HFUS and PA imaging, the LNA needs to achieve low noise and high bandwidth simultaneously. Conventionally, the LNA is designed as either a charge-sensitive amplifier (CSA) or a voltage amplifier. Although the CSA provides low noise performance ,9, the f2) cancellation. The constant transconductance, however, relies on precisely matched triode transistors.As the outputs of ultrasound transducers are normally in single-ended mode, the LNAs are typically limited to single-ended structures, thus suffering from even-order nonlinear distortion. To suppress the even-order harmonics, single-ended-to-differential conversion circuits have been utilized but with the penalties of additional noise and power consumption. To address this issue, a bulky LC resonator is used as a band-pass filter in  to rejecPower consumption is also a major concern in designing ultrasound LNAs. In beamforming applications where multiple channels are integrated on a single chip, the heat generated by the LNAs can significantly affect chip reliability. Low power consumption is also indispensable in portable ultrasound systems. To reduce LNA power consumption, a step-up balun with two secondary turns is developed in  to allev11), a voltage gain of 37 dB, and a THD of −55 dBc. The front-end consumes 37 mW of power from a 1.8 V supply and achieves a noise efficiency factor (NEF) of 2.66.In this article, which is an extension of ,17, a coThe overall LNA design was carried out by the following procedures. First, the typical electrical parameters of the PVDF ultrasound transducer for HFUS and PA imaging applications were obtained through experimental measurements. The parameters included the resonance frequency, output impedance, and output amplitude of the transducer. Then, an electrical model of the PVDF transducer was developed to facilitate the co-simulation of the LNA with the ultrasound transducer. Second, given the typical PVDF electrical parameters, the LNA design specifications, including gain and bandwidth, input impedance matching, and total harmonic distortion, were defined. Design solutions for improving the bandwidth, noise, linearity, and power efficiency of the LNA were then developed. A test chip was designed and fabricated in a 180 nm CMOS technology, and experimental measurements were carried out to validate the proposed LNA design solutions.The remainder of this paper is organized as follows. 2 transparent area at the center. As shown in Given that the typical resonance frequency of the PVDF transducer was about 50~80 MHz where the corresponding f−3dB bandwidth and wideband impedance matching.The resistive shunt-feedback amplifier, as depicted in M1 and assuming M1. The input impedance matching condition was then obtained asNeglecting the effect of parasitic capacitance X to node Y was derived asM1, respectively. Then, under the input impedance matched condition, the signal gain Next, we derived the signal gain of the amplifier M1. The noise factor of the amplifier [The major noise components of the amplifier were the thermal noises of mplifier  could be MOSFETs , and χ rM1. The main amplifier M1 had an inverting signal gain, as shown in Equation (4). The noise gain of the main amplifier M1 could be derived asVX,N denotes the input-referred thermal noise of M1, and VY,N is the corresponding output noise. From Equation (6), the main amplifier M1 had a non-inverting gain for noise. To cancel the noise of M1, the auxiliary amplifier, as shown in X. With the main amplifier and the auxiliary amplifier exhibiting opposite noise gains, noise cancellation can thus be achieved.To improve the noise performance, feedforward noise cancellation  was explM1 worked as the main amplifier, M2 with a cascode structure worked as the auxiliary amplifier, and M4 worked as a source follower combining the outputs of both amplifiers. The main amplifier and the auxiliary amplifier were connected in parallel with respect to node X. The input impedance condition of the LNA could be derived to be the same as in Equation (2).Z contributed by the main amplifier could be obtained asIgnoring the small gain reduction due to the source follower, the signal gain M2 and M4, respectively.The signal gain Z could be obtained asWith Equations (7)–(10), the noise-canceling condition at node M1 was canceled by the auxiliary amplifier M2. The input-referred noise of the overall amplifier was only determined by the auxiliary amplifier M2 and could be made small with a large Under the noise-canceling condition in Equation (11), the noise of the main amplifier MP in parallel with an NMOS-based resistive shunt-feedback amplifier MN. The drain current in each sub-amplifier as a function of the input signal was respectively obtained as [To suppress the second-order harmonics in the single-ended ultrasound LNA structure, complementary CMOS topology was investigated . As depiained as :(13)ids,By summing the two currents, the output current of the complementary amplifier was obtained asMP and a 120-μm/0.18-μm NMOS MN are plotted in From Equation (15), it can be observed that the second-order harmonic can be largely canceled if MP and MN transistors and could be obtained asWith the complementary CMOS topology, the input impedance matching condition was jointly determined by both the The signal gain of the complementary resistive shunt-feedback amplifier was derived asMP,CR and MN,CR, respectively, assuming MP,CR and MN,CR needed to be sized so that the output common-mode voltage was close to half VDD. The input common-mode voltage was set by the output common-mode voltage through the feedback resistor MP,CR and MN,CR were in saturation, which helped to further reduce the power consumption.To reduce the power consumption of the LNA, the current-reuse technique  was invef−3dB bandwidth of larger than 150 MHz. The use of a low VDD, however, mandated large sized transistors, especially in the auxiliary amplifiers, to maintain low noise performance. This led to a large parasitic capacitance at the amplifier input node, which could degrade the amplifier S11 at high frequencies. In designing the low-VDD current-reuse amplifier with feedforward noise-canceling, the VDD needs to be properly selected to tradeoff between noise, power saving, bandwidth, and high-frequency S11.Simulation results  showed tM1P and M1N are the main amplifiers, M2P and M2N are the auxiliary amplifiers, and M4P and M4N are the analog combiners, respectively. The sizes of the M1P and M1N transistors were 240-μm/0.18-μm and 120-μm/0.18-μm, respectively. Both transistors were biased with 1 mA current. The corresponding 2 and 2 and this helped to achieve the cancellation of the second-order harmonic distortion. The size of the M2P and M2N transistors were designed as 960-μm/0.18-μm and 480-μm/0.18-μm with the corresponding A resistive shunt-feedback LNA with the feedforward noise-canceling technique and the complementary topology was designed in a 180 nm CMOS technology. As shown in 11 and frequency response of the LNA, respectively. The S11 was better than −17 dB over the desired ultrasound transducer operation frequency range. The S11 at higher frequencies was slightly degraded by the main amplifier input node capacitance. The frequency response shows that the LNA had a gain of 19 dB, up to 120 MHz, in the typical corner and a f−3dB bandwidth of 770 MHz. The gain variation over the process, voltage, and temperatures (PVTs) was less than 2 dB.As shown in 11 and frequency response were measured with the Keysight N5247A network analyzer. The noise density and the THD were measured with the Keysight E4438C signal generator and the Keysight N9040B signal analyzer.A high-frequency ultrasound and photoacoustic imaging front-end, as shown in The Gain Method  has been11 was better than −16 dB over the frequency range of 30–120 MHz, as shown in f−3dB bandwidth of the front-end was 89 MHz. The noise performance of the front-end over 30–120 MHz is shown in IN, PP = 1 mV, the measured THD was better than −55 dBc over 30–80 MHz and was −51 dBc at 120 MHz.The measured SBW is the front-end bandwidth. The input-referred noise of the front-end over 30–120 MHz was 14.2 µV and the The noise efficiency factor , which cf−3dB bandwidth, and competitive power consumption, demonstrating the best NEF.f−3dB bandwidth and a wideband impedance matching. To mitigate the noise in the resistive shunt-feedback amplifier, a feedforward noise-canceling technique was developed. A complementary CMOS topology was also developed to cancel the amplifier’s second-order nonlinear distortion. An ultrasound receiver front-end, including the proposed LNA and a pseudo-differential VGA, was fabricated in a standard 180 nm CMOS process. Measured at 80 MHz, the front-end achieved an input-referred noise density of 1.36 nV/sqrt (Hz), a −16.4 dB input return loss, a 37 dB voltage gain, and a −55 dBc THD, while consuming 37 mW of power. The front-end demonstrated the best NEF with a large f−3dB bandwidth, wideband impedance matching, low noise and harmonic distortion, and competitive power consumption, making it suitable for high-frequency ultrasound transducer applications.This paper presents a low-noise amplifier front-end for high-frequency ultrasound transducers. The LNA employs a resistive shunt-feedback configuration to simultaneously achieve a large


Bacillus cereus group, Bacillus cytotoxicus, reliably harbors the coding gene of cytotoxin K-1 (CytK-1). This protein is a highly cytotoxic variant of CytK toxin, initially recovered from a diarrheal foodborne outbreak that caused the death of three people. In recent years, the cytotoxicity of B. cytotoxicus has become controversial, with some strains displaying a high cytotoxicity while others show no cytotoxicity towards cell lines. In order to better circumscribe the potential pathogenic role of CytK-1, knockout (KO) mutants were constructed in two B. cytotoxicus strains, E8.1 and E28.3. The complementation of the cytK-1 KO mutation was implemented in a mutant strain lacking in the cytK-1 gene. Using the tetrazolium salt (MTT) method, cytotoxicity tests of the cytK-1 KO and complemented mutants, as well as those of their wild-type strains, were carried out on Caco-2 cells. The results showed that cytK-1 KO mutants were significantly less cytotoxic than the parental wild-type strains. However, the complemented mutant was as cytotoxic as the wild-type, suggesting that CytK-1 is the major cytotoxicity factor in B. cytotoxicus.The thermotolerant representative of the  Bacillus cereus strain isolated from a food poisoning outbreak fatal to three elderly people in France in 1998. This B. cereus strain and its kin were later described as a new species, Bacillus cytotoxicus, the thermotolerant representative of the B. cereus group . This method assesses the cell’s mitochondrial activity as an indicator of cell viability and cytotoxicity, initially described by Mosmann . After ep < 0.05), a t-test  was performed with SPSS Statistics 26 .The normality of the data was investigated using the Kolmogorov–Smirnov test. Microsoft Excel 2016 was used to compute mean values and standard deviations  for each test condition. To determine whether the data were significantly different (